Novel fluorescent proteins

ABSTRACT

A GFP with an F64L mutation and an E222G mutation is provided. This GFP has a bigger Stokes shift compared to other GFPs making it very suitable for high throughput screening due to a better resolution. This GFP also has an excitation maximum between the yellow GFP and the cyan GFP allowing for cleaner band separation when used together with those GFPs.

[0001] This non-provisional application incorporates by reference thesubject matter of Application Nos. PA 2000 00953 and PA 2001 00739 filedin Denmark on Jun. 19, 2000 and May 10, 2001, respectively, on which apriority claim is based under 35 U.S.C. §119(a). This application alsoincorporates by reference the subject matter of co-pending U.S.Provisional Application Nos. 60/212,681 and 60/290,170 filed in theUnited States on Jun. 2, 2000 and May 9, 2001, respectively, on which apriority claim is based under 35 U.S.C. §119(e).

FIELD OF INVENTION

[0002] The present invention relates to novel variants of thefluorescent protein GFP having improved fluorescence properties.

BACKGROUND

[0003] The discovery that Green Fluorescent Protein (GFP) from thejellyfish A. victoria retains its fluorescent properties when expressedin heterologous cells has provided biological research with a new,unique and powerful tool (Chalfie et al (1994). Science 263:802; Prasher(1995) Trends in Genetics 11:320; WO 95/07463). A very important aspectof using recombinant, fluorescent proteins in studying cellularfunctions is the non-invasive nature of the assay. This allows detectionof cellular events in intact, living cells.

[0004] The excitation spectrum of the green fluorescent protein fromAequorea victoria shows two peaks: A major peak at 396 nm, which is inthe potentially cell damaging UV range, and a lesser peak at 475 nm,which is in an excitation range that is much less harmful to cells.

[0005] To improve the wild type GFP, a range of mutations have beendescribed. Heim (GFP (Heim et al. (1994). Proc.Natl.Acad.Sci. 91:12501)described the discovery of a blue fluorescent variant which has greatlyincreased the potential applications of using fluorescent recombinantprobes to monitor cellular events or functions, since the availabilityof probes having different excitation and emission spectra permitssimultaneous monitoring of more than one process. However, the bluefluorescing variant described by Heim et al, Y66H-GFP, suffers fromcertain limitations: The blue fluorescence is weak (emission maximum at448 nm), thus making detection difficult, and necessitating prolongedexcitation of cells expressing Y66H-GFP. Moreover, the prolonged periodof excitation is damaging to cells especially because the excitationwavelength is in the UV range, 360 nm-390 nm.

[0006] Heim et al.(1995), Nature, Vol. 373, p. 663-4, discloses aSer65Thr mutation of GFP (S65T) having longer wavelengths of excitationand emission, 490 nm and 510 nm, respectively, than the wild-type GFPand wherein the fluorophore formation proceeded about fourfold morerapidly than in the wild-type GFP.

[0007] Ehrig et al. (1995) FEBS Letters 367,163-166, discloses a E222Gmutant of the Aequorea green fluorescent protein. This mutation has anexcitation maximum of 481 nm and an emission maximum at 506 nm.

[0008] Expression of GFP or its fluorescent variants in living cellsprovides a valuable tool for studying cellular events and it is wellknown that many cells, including mammalian cells, are incubated atapproximately 37° C. in order to secure optimal and/or physiologicallyrelevant growth. Cell lines originating from different organisms ortissues may have different relevant temperatures ranging from about 35°C. for fibroblasts to about 38° C.-39° C. for mouse p-cells. Experiencehas shown, however, that the fluorescent signal from cells expressingGFP is weak or absent when said cells are incubated at temperaturesabove room temperature, cf. Webb, C. D. et al., Journal of Bacteriology,Oct.1995, p. 5906-5911. Ogawa H. et al., Proc. Natl. Acad. Sci. USA,Vol. 92, pp.11899-11903, December 1995, and Lim et al. J. Biochem.118,13-17 (1995). The improved fluorescent variant S65T described by Heim etal. (1995) supra also displays very low fluorescence when incubatedunder normal culture conditions (37° C.), cf. Kaether and Gerdes FEBSLetters 369 (1995) pp. 267-271. Many experiments involving the study ofcell metabolism are dependent on the possibility of incubating the cellsat physiologically relevant temperatures, i.e. temperatures at about 37°C.

[0009] Thastrup et al. (1997) EP 0 851 874 describes fluorescentproteins that exhibit high fluorescence in cells expressing them whensaid cells are incubated at a temperature of 30° C. or above. This isobtained with the amino acid in position 1 preceding the chromophore hasbeen mutated. Examples of such mutations are F64L, F64I, F64V F64A andF64G.

[0010] Various authors have experimented with combinations of mutations.One such combination is the F64L, S65T GFP (EGFP). EGFP exhibits highfluorescence when expressed at 30° C. or above and has an excitationmaximum at 488 nm.

SUMMARY OF THE INVENTION

[0011] The present invention provides novel fluorescent proteins, suchas F64L-E222G-GFP that result in a cellular fluorescence far exceedingthe cellular fluorescence when expressed at 37° C. and when excitated at450 to 500 nm compared to the parent proteins, i.e. GFP, the bluevariant Y66H-GFP the S65T-GFP variant, and F64L-GFP. This greatlyimproves the usefulness of fluorescent proteins in studying cellularfunctions in living cells.

[0012] It is shown that GFP mutated at the 64 position from F to L(F64L) and at the 222 position from E to G (E222G) has remarkableproperties. It is first shown that the F64L,E222G-GFP has an entirelydifferent spectrum than F64L,S65T-GFP (Example 2). In contrast, there isno substantial difference between folding characteristics (measured asthe time when fluorescence is observed between the two GFPs, Example 3).Likewise, there was no difference between the pH sensitivity of the twoGFPs (Example 4). The observed brightness of the E222G versus the S65Tmutated F64L-GFPs is dependent on the test conditions (Example 5).

DETAILED DESCRIPTION OF THE INVENTION

[0013] One aspect of the present invention relates to a fluorescentprotein derived from Green Fluorescent Protein (GFP) or any functionalGFP analogue, wherein the amino acid in position 1 preceding thechromophore has been mutated and wherein the Glutamic acid in position222 has been mutated said mutated GFP has an excitation maximum at ahigher wavelength compared to F64L-GFP and the fluorescence is increasedwhen the mutated GFP is expressed in cells incubated at a temperature of30° C. or above compared to wild-type GFP.

[0014] The excitation and emission characteristics of the F64L,E222G-GFPdiffer significantly from wild-type GFP and EGFP. Existing fluorescentproteins have demonstrated utility for research applications such asquantitative fluorescence microscopy (Patterson, G. H., et al (1997).Biophysical J. 73:2782-2790; Piston, D. W.,et al (1999) Meth. Cell Biol.58:31-48). It is now clear, however, that the optimal fluorescentprotein characteristics for high-throughput screening (HTS) applicationsin drug discovery differ somewhat from those for research applications(Kain, S. R. (1999) Drug Discovery Today 4:304-312). For example,factors such as optimal and signal/noise are more important for HTSapplications in drug discovery than are absolute brightness of probessuch as fluorescent proteins. The F64L,E222G-GFP described in thispatent application has an excitation maximum of 470 nm and an emissionmaximum of 505 nm (see FIG. 3:), compared to the respective excitationand emission maxima of 490 nm and 510 nm for EGFP. This results in aStokes shift of 35 nm for F64L,E222G-GFP, as compared to 20 nm for EGFP.This results in a significant increase in the excitation-emission bandseparation for F64L,E222G-GFP relative to EGFP with several implicationsfor the use of F64L,E222G-GFP in high-throughput screening. Some ofthese are listed below:

[0015] 1. The increased Stokes shift of F64L,E222G-GFP results inincreased spectral resolution of its excitation and emission peaks. Thisenables more complete band separation using a conventional dichroicbeam-splitter, and decreased background signal for assays incorporatingF64L,E222G-GFP relative to assays based on EGFP.

[0016] 2. F64L,E222G-GFP fluorescence can be excited by conventionallight sources using narrow band filters, or commercially available laserproducing lines at 472 nm. In either case, the greater Stokes shift ofF64L,E222G-GFP results in lower cross-talk from excitation light to thetoe of the emission spectrum.

[0017] 3. The excitation maximum of F64L,E222G-GFP falls midway betweenthose of the cyan fluorescent protein variant (ECFP, excitation max ˜433nm) and the yellow fluorescent protein variant (EYFP, excitation max˜513 nm). Because of this, it will allow for cleaner band separationwhen used together with those probes, and it is optimized for assayapplications in which several GFP-labeled components will bemultiplexed.

[0018] Many sources of GFPs exist. Examples are GFP derived fromAequorea victoria and GFP derived from Renilla. Various GFPs have beenisolated from Renilla examples are reniformis and mulleri. As describedin the examples and in SEQ ID NOs: 3 and 4, the chromophore in Aequoreavictoria is in position 65-67 of the predicted primary amino acidsequence of GFP. Thus, in a preferred embodiment the GFP is derived fromAequorea victoria.

[0019] It is preferred that the mutation at F64 is a mutation to analiphatic amino acid. Examples are F64L, F64I, F64V, F64A, and F64G,wherein the F64L substitution being most preferred. However othermutations, e.g. deletions, insertions, or post-translationalmodifications immediately preceding the chromophore are also included inthe invention, provided that they result in improved fluorescenceproperties of the various fluorescent proteins. It should be noted thatextensive deletions may result in loss of the fluorescent properties ofGFP.

[0020] The E222G, E222A, E222V, E222L, E222I, E222F, E222S, E222T,E222N, E222Q substitutions are preferred, the E222G substitution (thatis substitution to Glycine) being most preferred.

[0021] A preferred sequence of the gene encoding GFP derived fromAequorea victoria is disclosed in SEQ ID NO: 3 (enhanced) and in SEQ IDNO: 7 (jelly fish). SEQ ID NO: 1 shows the nucleotide sequence ofF64L-GFP with humanised codon. SEQ ID NO: 5 shows the nucleotidesequence of F64L-GFP with jellyfish codon. Besides, the novelfluorescent proteins may also be derived from other fluorescent proteinsas mentioned above.

[0022] Herein the abbreviations used for the amino acids are thosestated in J. Biol. Chem. 243 (1968), 3558.

[0023] One aspect of the invention relates to a nucleotide sequencecoding for the Fluorescent protein F64L-E222G-GFP. An example of suchF64L-E222G-GFP is shown in list 2. In a preferred aspect the nucleotidesequence is in the form of a DNA sequence.

[0024] The DNA construct of the invention encoding the novel fluorescentproteins may be prepared synthetically by established standard methods,e.g. the phosphoamidite method described by Beaucage and Caruthers,Tetrahedron Letters 22 (1981), 1859-1869, or the method described byMatthes et al., EMBO Journal 3 (1984), 801-805. According to thephosphoamidite method, oligonucleotides are synthesized, e.g. in anautomatic DNA synthesizer, purified, annealed, ligated and cloned insuitable vectors.

[0025] The DNA construct may also be prepared by polymerase chainreaction (PCR) using specific primers, for instance as described in U.S.Pat. No. 4,683,202 or Saiki et al., Science 239 (1988), 487-491. A morerecent review of PCR methods may be found in PCR Protocols, 1990,Academic Press, San Diego, Calif., USA.

[0026] The DNA construct of the invention may be inserted into arecombinant vector which may be any vector which may conveniently besubjected to recombinant DNA procedures. The choice of vector will oftendepend on the host cell into which it is to be introduced. Thus, thevector may be an autonomously replicating vector, i.e. a vector whichexists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g. a plasmid. Alternatively,the vector may be one which, when introduced into a host cell, isintegrated into the host cell genome and replicated together with thechromosome(s) into which it has been integrated.

[0027] The vector is preferably an expression vector in which the DNAsequence encoding the fluorescent protein of the invention is operablylinked to additional segments required for transcription of the DNA. Ingeneral, the expression vector is derived from plasmid or viral DNA, ormay contain elements of both. The term, “operably linked” indicates thatthe segments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in a promoter andproceeds through the DNA sequence coding for the fluorescent protein ofthe invention.

[0028] The promoter may be any DNA sequence which shows transcriptionalactivity in the host cell of choice and may be derived from genesencoding proteins either homologous or heterologous to the host cell,including native Aequorea GFP genes.

[0029] Examples of suitable promoters for directing the transcription ofthe DNA sequence encoding the fluorescent protein of the invention inmammalian cells are the SV40 promoter (Subramani et al., Mol. Cell Biol.1 (1981), 854 -864), the MT-1 (metallothionein gene) promoter (Palmiteret al., Science 222 (1983), 809-814) or the adenovirus 2 major latepromoter.

[0030] An example of a suitable promoter for use in insect cells is thepolyhedrin promoter (U.S. Pat. No. 4,745,051; Vasuvedan et al., FEBSLett. 311, (1992) 7-11), the P10 promoter (J. M. Vlak et al., J. Gen.Virology 69, 1988, pp. 765-776), the Autographa californica polyhedrosisvirus basic protein promoter (EP 397 485), the baculovirus immediateearly gene 1 promoter (U.S. Pat. No. 5,155,037; U.S. Pat. No.5,162,222), or the baculovirus 39K delayed-early gene promoter (U.S.Pat. No. 5,155,037; U.S. Pat. No. 5,162,222).

[0031] Examples of suitable promoters for use in yeast host cellsinclude promoters from yeast glycolytic genes (Hitzeman et al., J. Biol.Chem. 255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1(1982), 419-434) or alcohol dehydrogenase genes (Young et al., inGenetic Engineering of Microorganisms for Chemicals (Hollaender et al,eds.), Plenum Press, New York, 1982), or the TPI1 (U.S. Pat. No.4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652-654)promoters.

[0032] Examples of suitable promoters for use in filamentous fungus hostcells are, for instance, the ADH3 promoter (McKnight et al., The EMBO J.4 (1985), 2093-2099) or the tpiA promoter. Examples of other usefulpromoters are those derived from the gene encoding A. oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, A. niger neutralα-amylase, A. niger acid stable α-amylase, A. niger or A. awamoriglucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkalineprotease, A. oryzae triose phosphate isomerase or A. nidulansacetamidase. Preferred are the TAKA-amylase and gluA promoters.

[0033] Examples of suitable promoters for use in bacterial host cellsinclude the promoter of the Bacillus stearothermophilus maltogenicamylase gene, the Bacillus licheniformis alpha-amylase gene, theBacillus amyloliquefaciens BAN amylase gene, the Bacillus subtilisalkaline protease gene, or the Bacillus pumilus xylosidase gene, or bythe phage Lambda P_(R) or P_(L) promoters or the E. coli lac, trp or tacpromoters.

[0034] The DNA sequence encoding the novel fluorescent proteins of theinvention may also, if necessary, be operably connected to a suitableterminator, such as the human growth hormone terminator (Palmiter etal., op. cit.) or (for fungal hosts) the TPI1 (Alber and Kawasaki, op.cit.) or ADH3 (McKnight et al., op. cit.) terminators. The vector mayfurther comprise elements such as polyadenylation signals (e.g. fromSV40 or the adenovirus 5 Elb region), transcriptional enhancer sequences(e.g. the SV40 enhancer) and translational enhancer sequences (e.g. theones encoding adenovirus VA RNAs).

[0035] The recombinant vector may further comprise a DNA sequenceenabling the vector to replicate in the host cell in question. Anexample of such a sequence (when the host cell is a mammalian cell) isthe SV40 origin of replication.

[0036] When the host cell is a yeast cell, suitable sequences enablingthe vector to replicate are the yeast plasmid 2 μ replication genes REP1-3 and origin of replication.

[0037] The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the genecoding for dihydrofolate reductase (DHFR) or the Schizosaccharomycespombe TPI gene (described by P. R. Russell, Gene 40, 1985, pp. 125-130),or one which confers resistance to a drug, e.g. ampicillin, kanamycin,tetracyclin, chloramphenicol, neomycin or hygromycin. For filamentousfungi, selectable markers include amdS, pyrG, argB, niaD, sC.

[0038] The procedures used to ligate the DNA sequences coding for thefluorescent protein of the invention, the promoter and optionally theterminator and/or secretory signal sequence, respectively, and to insertthem into suitable vectors containing the information necessary forreplication, are well known to persons skilled in the art (cf., forinstance, Sambrook et al., op.cit.).

[0039] The host cell into which the DNA construct or the recombinantvector of the invention is introduced may be any cell which is capableof expressing the present DNA construct and includes bacteria, yeast,fungi and higher eukaryotic cells.

[0040] Examples of bacterial host cells which, on cultivation, arecapable of expressing the DNA construct of the invention aregrampositive bacteria, e.g. strains of Bacillus, such as B. subtilis, B.licheniformis, B. lentus, B. brevis, B. stearothermophilus, B.alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B.lautus, B. megatherium or B. thuringiensis, or strains of Streptomyces,such as S. lividans or S. murinus, or gramnegative bacteria such asEcherichia coli. The transformation of the bacteria may be effected byprotoplast transformation or by using competent cells in a manner knownper se (cf. Sambrook et al., supra).

[0041] Examples of suitable mammalian cell lines are the HEK293 and theHeLa cell lines, primary cells, and the COS (e.g. ATCC CRL 1650), BHK(e.g. ATCC CRL 1632, ATCC CCL 10), CHL (e.g. ATCC CCL39) or CHO (e.g.ATCC CCL 61) cell lines. Methods of transfecting mammalian cells andexpressing DNA sequences introduced in the cells are described in e.g.Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg,J. Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl. Acad.Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaroand Pearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb,Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845.

[0042] Examples of suitable yeast cells include cells of Saccharomycesspp. or Schizosaccharomyces spp., in particular strains of Saccharomycescerevisiae or Saccharomyces kluyveri. Methods for transforming yeastcells with heterologous DNA and producing heterologous polypeptidestherefrom are described, e.g. in U.S. Pat. No. 4,599,311, U.S. Pat. No.4,931,373, U.S. Pat. Nos. 4,870,008, 5,037,743, and U.S. Pat. No.4,845,075, all of which are hereby incorporated by reference.Transformed cells are selected by a phenotype determined by a selectablemarker, commonly drug resistance or the ability to grow in the absenceof a particular nutrient, e.g. leucine. A preferred vector for use inyeast is the POT1 vector disclosed in U.S. Pat. No. 4,931,373. The DNAsequence encoding the fluorescent protein of the invention may bepreceded by a signal sequence and optionally a leader sequence , e.g. asdescribed above. Further examples of suitable yeast cells are strains ofKluyveromyces, such as K. lactis, Hansenula, e.g. H. polymorpha, orPichia, e.g. P. pastoris (cf. Gleeson et al., J. Gen. Microbiol. 132,1986, pp. 3459-3465; U.S. Pat. No. 4,882,279).

[0043] Examples of other fungal cells are cells of filamentous fungi,e.g. Aspergillus spp., Neurospora spp., Fusarium spp. or Trichodermaspp., in particular strains of A. oryzae, A. nidulans or A. niger. Theuse of Aspergillus spp. for the expression of proteins is described in,e.g., EP 272 277, EP 230 023, EP 184 438.

[0044] When a filamentous fungus is used as the host cell, it may betransformed with the DNA construct of the invention, conveniently byintegrating the DNA construct in the host chromosome to obtain arecombinant host cell. This integration is generally considered to be anadvantage as the DNA sequence is more likely to be stably maintained inthe cell. Integration of the DNA constructs into the host chromosome maybe performed according to conventional methods, e.g. by homologous orheterologous recombination.

[0045] Transformation of insect cells and production of heterologouspolypeptides therein may be performed as described in U.S. Pat. No.4,745,051; U.S. Pat. No. 4,879,236; U.S. Pat. No. 5,155,037; 5,162,222;EP 397,485) all of which are incorporated herein by reference. Theinsect cell line used as the host may suitably be a Lepidoptera cellline, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf.U.S. Pat. No. 5,077,214). Culture conditions may suitably be asdescribed in, for instance, WO 89/01029 or WO 25 89/01028, or any of theaforementioned references.

[0046] One aspect of the invention relates to a host transformed with aDNA construct according to any of the preceding aspects. The transformedor transfected host cell described above is then cultured in a suitablenutrient medium under conditions permitting the expression of thepresent DNA construct after which the cells may be used in the screeningmethod of the invention. Alternatively, the cells may be disrupted afterwhich cell extracts and/or supernatants may be analysed forfluorescence.

[0047] The medium used to culture the cells may be any conventionalmedium suitable for growing the host cells, such as minimal or complexmedia containing appropriate supplements. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedrecipes (e.g. in catalogues of the American Type Culture Collection).

[0048] In the method of the invention, the fluorescence of cellstransformed or transfected with the DNA construct of the invention maysuitably be measured in a spectrometer or a fluorescence microscopewhere the spectral properties of the cells in liquid culture may bedetermined as scans of light excitation and emission.

[0049] One aspect of the invention relates to a fusion compoundconsisting of a fluorescent protein (F64L-E222G-GFP ), wherein the(F64L-E222G-GFP ) is linked to a polypeptide. Examples of suchpolypeptide is kinase, preferably the catalytic subunit of proteinkinase A, or protein kinase C, or Erk1, or a cytoskeletal element.

[0050] The invention further relates to a process for preparing apolypeptide, comprising cultivating a host according to any of thepreceding aspects and obtaining therefrom the polypeptide expressed bysaid nucleotide sequence.

[0051] The various aspects of the invention have a plethora of uses.Some of these are described below:

[0052] Use of F64L-E222G-GFP in an in vitro assay for measuring proteinkinase activity, or dephosphorylation activity, or for measuring proteinredistribution.

[0053] Use of F64L-E222G-GFP as a protein tag in living and fixed cells.Due to the strong fluorescence the novel proteins are suitable tags forproteins present at low concentrations. Since no substrate is needed andvisualisation of the cells does not damage the cells dynamic analysiscan be performed.

[0054] Use as an organelle tag. More than one organelle can be taggedand visualised simultaneously in living cells, e.g. the endoplasmicreticulum and the cytoskeleton.

[0055] Use as a secretion marker. By fusion of F64L-E222G-GFP to asignal peptide or a peptide to be secreted, secretion may be followedon-line in living cells. A precondition for that is that the maturationof a detectable number of novel fluorescent protein molecules occursfaster than the secretion.

[0056] Use as genetic reporter or protein tag in transgenic animals. Dueto the strong fluorescence of the novel proteins, they are suitable astags for proteins and gene expression, since the signal to noise ratiois significantly improved over the prior art proteins, such as wild-typeGFP.

[0057] Use as a cell or organelle integrity marker. By co-expressing twoof the novel proteins, the one targeted to an organelle and the otherexpressed in the cytosol, it is possible to calculate the relativeleakage of the cytosolic protein and use that as a measure of cellintegrety.

[0058] Use as a marker for changes in cell morphology. Expression of thenovel proteins in cells allows easy detection of changes in cellmorphology, e.g. blebbing, caused by cytotoxic agents or apoptosis. Suchmorphological changes are difficult to visualize in intact cells withoutthe use of fluorescent probes.

[0059] Use as a transfection marker, and as a marker to be used incombination with FACS sorting. Due to the increased brightness of thenovel proteins the quality of cell detection and sorting can besignificantly improved.

[0060] Use as real-time probe working at near physiologicalconcentrations Since F64L-E222G-GFP is significantly brighter than wildtype GFP and F64L-GFP when expressed in cells at about 37° C. andexcited with light at about 490 nm, the concentration needed forvisualization can be lowered. Target sites for enzymes engineered intothe novel proteins, e.g. F64L-E222G-GFP, can therefore be present in thecell at low concentrations in living cells. This is important for tworeasons: 1) The probe must interfere as little as possible with theintracellular process being studied; 2) the translational andtranscriptional apparatus should be stressed minimally.

[0061] The novel proteins can be used as reporters to monitor live/deadbiomass of organisms, such as fungi. By constitutive expression ofF64L-E222G-GFP in fungi the viable biomass will light up.

[0062] Transposon vector mutagenesis can be performed using the novelproteins as markers in transcriptional and translational fusions.

[0063] Transposons to be used in microorganisms encoding the novelproteins. The transposons may be constructed for translational andtranscriptional fusions. To be used for screening for promoters.

[0064] Transposon vectors encoding the novel proteins, such asF64L-E222G-GFP, can be used for tagging plasmids and chromosomes.

[0065] Use as a reporter for bacterial detection by introducing thenovel proteins into the genome of bacteriophages.

[0066] By engineering the novel proteins, e.g. F64L-E222G-GFP, into thegenome of a phage a diagnostic tool can be designed. F64L-E222G-GFP willbe expressed only upon transfection of the genome into a living host.The host specificity is defined by the bacteriophage.

[0067] The invention is further illustrated in the following exampleswith reference to the appended sequence lists. TABLE 1 List of sequencesNucleotide SEQ ID Protein SEQ ID Name NO: NO: e-F64L-GFP 1 2 (PS399)e-FG4L-E222G- 3 4 GFP (PS699) jf-F64L-GFP 5 6 (PS350) jf-F64L-E222G- 7 8GFP (PS1186)

LEGEND TO FIGURES

[0068] PS codes are explained in Table 2.

[0069]FIG. 1 Excitation spectra of PS1189 (excitation maximum at 492nm), PS1191 (excitation maximum at 468 nm), PS1185 (excitation maximumat 490 nm) and PS1186 (excitation maximum at 473 nm). The emissions wererecorded at 560 nm. The samples of PS1189 and PS1191 were 2-fold dilutedand the samples of PS1185 and PS1186 were 10-fold diluted.

[0070]FIG. 2 Emission spectra of PS1189 (emission maximum at 509 nm),PS1191 (emission maximum at 505 nm), PS1185 (emission maximum at 510 nm)and PS1186 (emission maximum at 506 nm). Excitation was at 430 nm. Thesamples of PS1189, PS1191 and PS1185 were 2-fold diluted and the sampleof PS1186 was 10-fold diluted. The curves for PS1189 and PS1191 relateto the primary y-axis whereas the curves for PS1185 and PS1186 relate tothe secondary y-axis.

[0071]FIG. 3 Overlapping excitation (Ex) and emission (Em) spectra ofPS1189 (panel A), PS1191 (panel B), PS1185 (panel C), and PS1186 (panelD). The excitation curve to the left and the excitation curve to theright relate to the primary and secondary y-axis, respectively.

[0072]FIG. 4 This figure shows the images collected after Lipofectamine2000 transfection. eF64L,E222G (PS699) is at the top of the right columnreferred to as E222G, eF64L,S65T-GFP (PS279) is at the top of the leftcolumn referred to as EGFP.

[0073]FIG. 5 Comparing the pH sensitivity of EGFP (PS279) andeF64L,E222G-GFP (PS699).

EXAMPLES Example 1 Construction of GFP Plasmids

[0074] Plasmids pEGFP-N1 (GenBank accession number U55762) and pEGFP-C1(GenBank accession number U55763) both contain a derivative of GFP inwhich one extra amino acid has been added at position two to provide abetter translational start sequence (a Kozak sequence) and so the totalnumber of amino acids is increased by one to 239 instead of the 238found in wildtype GFP. Therefore the denomination of mutations in GFP inthese plasmids strictly should be referred to as e.g. F65L rather thanF64L. However, to avoid this source of confusion and because the GFPcommunity has adopted the numbering system of wildtype GFP in itscommunications, the numbers used here conform to the commonly usednaming of mutations in wildtype GFP. The relevant mutations in thisrespect are F64L, S65T, and E222G.

[0075] Plasmids pEGFP-N1 and pEGFP-C1 contain the following mutations inthe chromophore: F64L and S65T. The codon usage of the GFP DNA sequencehas been optimized for expression in mammalian cells. N1 and C1 refer tothe position of multiple cloning sites relative to the GFP sequence.

[0076] To construct a plasmid combining F64L and E222G, pEGFP-N1 andpEGFP-C1 were first subjected to PCR with primers 9859 and 9860described below. The primers are complementary to the DNA sequencearound the chromophore region and introduce a point mutation changingthe threonine at position 65 to serine. In addition the primersintroduce a unique Spe1 restriction site by silent mutation. The 4.7 kbPCR products were digested with Spe1, religated, and transformed intoE.coli. The resulting plasmids are referred to as PS399 (N1 context) andPS401 (C1 context). These plasmids contain the chromophore sequence64-LSYG-67. Plasmids PS399 and PS401 were subjected to Quick-Changemutagenesis (Stratagene) employing PCR with primers 0225 and 0226described below. These primers are complementary to sequences near theC-terminus of the GFP and change glutamate at position 222 to glycine,and in addition they introduce an Avr2 restriction site by silentmutation. The resulting plasmids are referred to as PS699 (N1 context)and PS701 (C1 context). They combine an LSYG chromophore with E222G withhumanised codon and is referred to as eF64L,E222G (see sequence list 2)9859-top: 5′-TGTACTAGTGACCACCCTGTCTTACGGCGTGCA-3′ 9860-bottom:5′-CTGACTAGTGTGGGCCAGGGCACGGGCAGC-3′ 0225-bottom:5′-CCCGGCGGCGGTCACGAACCCTAGGAGGACCATGTGATCGCG-3′ 0226-top:5′-CGCGATCACATGGTCCTCCTAGGGTTCGTGACCGCCGCCGGG-3′

[0077] A plasmid encoding a GFP directly derived from jellyfish withF64L (disclosed in FIG. 4 of WO97/11094,) was subjected to PCR withprimers 9840 & 9841 described below. The PCR product was digested withrestriction enzymes Age1 and Acc65 and ligated into pEGFP-N1 digestedwith Age1 and BsrG1. This replaces EGFP with F64L-GFP and introduces anamino acid change L236G near the c-terminus as a consequence of joiningAcc65 and BsrG1 sites. This plasmid is referred to as PS350.

[0078] A plasmid encoding a GFP directly derived from jellyfish withF64L, S65T (disclosed in FIG. 5 of WO97/11094,) was subjected to PCRwith primers 9840 & 9841 described below. The PCR product was digestedwith restriction enzymes Age1 and Acc65 and ligated into pEGFP-N1digested with Age1 and BsrG1. This replaces EGFP with F64L, S65T-GFP andintroduces an amino acid change L236G near the c-terminus as aconsequence of joining Acc65 and BsrG1 sites. This plasmid is referredto as PS351.

[0079] Plasmid PS350 was subjected to QuickChange PCR (Stratagene) withprimers 0317 & 0318 described below. This introduces E222G by mutationand an Avr2 restriction site by silent mutation. This plasmid isreferred to as PS832.

[0080] Plasmid PS832 was subjected to QuickChange PCR (Stratagene) withprimers 0325 & 0326 described below. This introduces L64F by mutationand a Psp1406 restriction site by silent mutation. This plasmid isreferred to as PS845.

[0081] A plasmid encoding a GFP directly derived from jellyfish(disclosed in FIG. 2a of WO97/11094) was subjected to PCR with primers9840 & 9841 described below. The PCR product was digested withrestriction enzymes Age1 and Acc65 and ligated into pEGFP-N1 digestedwith Age1 and BsrG1. This replaces EGFP with wildtype GFP and introducesan amino acid change L236G near the c-terminus as a consequence ofjoining Acc65 and BsrG1 sites. This plasmid is referred to as PS854.

[0082] Plasmid PS399 was subjected to QuickChange PCR (Stratagene) withprimers 0327 & 0328 described below. This introduces L64F by mutationand a Psp1406 restriction site by silent mutation. This plasmid isreferred to as PS844.

[0083] Plasmid PS699 was subjected to QuickChange PCR (Stratagene) withprimers 0327 & 0328 described below. This introduces L64F by mutationand a Psp1406 restriction site by silent mutation. This plasmid isreferred to as PS846. 9840-top: 5′-GTACCGGTCACCATGAGTAAAGGAGAAGAAC-3′9841-bottom: 5′-TTATTGGTACCCTTCATCCATGCCATGTG-3′ 0317-top:5′-GAGATCACATGATCCTCCTAGGGTTTGTAACAGCTGCTGGG-3′ 0318-bottom:5′-CCCAGCAGCTGTTACAAACCCTAGGAGGATCATGTGATCTC-3, 0325-top:5′-CCAACGCTTGTCACAACGTTTTCTTATGGTGTTC-3′ 0326-bottom:5′-GAACACCATAAGAAAACGTTGTGACAAGCGTTGG-3′ 0327-top:5′-CCCACACTAGTGACAACGTTTTCTTACGGCGTGC-3′ 0328-bottom:5′-GCACGCCGTAAGAAAACGTTGTCACTAGTGTGGG-3′

[0084] Plasmids encoding GFPs in jellyfish codon context (PS350, PS351,PS832, PS845, PS854) were subjected to PCR with primers 1259 and 1260described below. The ca 0.8 kb PCR products were cut with restrictionenzymes BspH1 and BamH1, and ligated into E.coli expression vectorpTrcHis (from Invitrogen) cut with Nco1 and BamH1. This places the GFPsunder control of the ITPG-inducible promoter in the vector. The bottomprimer 1260 also changes the glycine at position 236 back to leucine.The resulting plasmids are referred to as PS1184 (jf-F64L-GFP), PS1185(jf-F64L,S65T-GFP), PS1186 (jf-F64L,E222G-GFP), PS1187 (jf-E222G-GFP)and PS (jf-GFP).

[0085] Plasmids encoding GFPs in humanised enhanced codon context(PS279=pEGFP-N1 (Clontech), PS399, PS699, PS844, PS846) were subjectedto PCR with primers 1261 and 1262 described below. The ca 0.8 kb PCRproducts were cut with restriction enzymes Nco1 and BamH1, and ligatedinto E.coli expression vector pTrcHis (from Invitrogen) cut with Nco1and BamH1. This places the GFPs under control of the ITPG-induciblepromoter in the vector. The resulting plasmids are referred to as PS1189(e-F64L,S65T-GFP=EGFP), PS1190 (e-F64L-GFP), PS1191 (e-F64L,E222G-GFP),PS1192 (e-GFP) and PS1193 (e-E222G-GFP). 1259-top:5′-GTTGTTTCATGAGTAAAGGAGAAGAACTTTTC-3′ 1260-bottom:5′-GTTGGATCCTTATTTGTATAGTTCATCCATG-3′ 1261-top.5′-GTTGTTCCATGGTGAGCAAGGGCGAGGAGCTG- 3′ 1262-bottom:5′-GTTGGATCCTTACTTGTACAGCTCGTCCATG-3′

[0086] The plasmids described above were transformed into E.coli strainDH5alpha (Life Technologies). Single colonies were picked and grownovernight at 37C in LB medium containing 1 mM IPTG. 0.5 ml cells werepelleted and stored at −20C until they were analyzed. TABLE 2 Summarytable of plasmids encoding GFPs with indicated amino acids at positions64, 65 and 222. mammalian Back- E. coli cell ex- bone- aa aa aa expres-pression codon us- pos pos pos sion plasmid age 64 65 222 plasmid PS846e-E222G-GFP enhanced F S G PS1193 PS844 e-GFP enhanced F S E PS1192PS699 e-F64L,E222G- enhanced L S G PS1191 GFP PS399 e-F64L-GFP enhancedL S E PS1190 PS279 EGFP enhanced L T E PS1189 PS854 jf-GFP jellyfish F SE P51188 PS845 jf-E222G-GFP jellyfish F S G PS1187 PS832 jf-F64L,E222G-jellyfish L S G PS1186 GFP PS351 jf-F64L,S65T- jellyfish L T E PS1185GFP PS350 jf-F64L-GFP jellyfish L S E PS1184

Example 2 Determination of Spectral Properties of Proteins EGFP andeF64L,E222G

[0087] Plasmids expressing EGFP from plasmid pEGFP-N1 (also referred toas PS279), and eF64L,E222G from plasmid PS699 were transfected intoE.Coli TOP10 cells (Invitrogen) using lipofectamine 2000 (from LifeTechnologies) according to manufacturers recommendations. After 5 dayscells were collected and resuspended in extraction buffer 50 mM TRIS(pH8.0) with 1 mM DTT. Cells were lysed by 3 cycles of freeze-thaw. Celldebris was centrifuged out at 10000 g in acooled centrifuge. NaCl wasadded to 100 mM.

[0088] The cell pellets were resuspended in 1000 μl of H₂O each (2-folddilution relative to volumes of pelleted cultures) and transferred to1.0×0.5 cm plastic cuvettes and the following excitation and emissionspectra were recorded on a Perkin Elmer LS50B luminescence spectrometer:Excitation spectrum: Excitation at 350-525 nm (5 nm slit width) Emission560 nm (10 nm slit width) Data presented in FIG. 1. Emission spectrum:Excitation at 430 nm (10 nm slit width) Emission 450-550 nm (5 nm slitwidth) Data presented in FIG. 2.

[0089] Using the same settings, excitation and emission spectra of10-fold (200 μl of 2-fold diluted cells mixed with 800 μl of water)diluted cells were recorded for the strongly fluorescent samplesexpressed from cDNAs with jellyfish backbone (PS1185 and PS1186).

[0090] In contrast to the expression levels, the fluorescence propertiesof the probes were independent of the codon usage. The spectra recordedfor the probes with Thr65:E222 (PS1185 and PS1189) were very similar(excitation and emission maxima at 490-492 nm and 509-510 nm,respectively) and with Stokes shifts of 17-20 nm. Likewise, the spectrarecorded for the probes with Ser65:G222 (PS1186 and PS1191) were verysimilar (excitation and emission maxima at 468-473 nm and 505-506 nm,respectively) and with Stokes shifts of 33-37 nm.

Example 3 Determination of Time to Fluorescence of EGFP and eF64L,E222Gin CHO Cells

[0091] Three, 2 well chambers with CHOhIR cells were transfected withplasmid PS279 expressing EGFP and plasmid PS699 expression eF64L,E222Gusing the Lipofectamine transfection method.

[0092] Fluorescence from the cells was checked at regular intervalsafter transfection.

[0093] Lipofectamine 2000 transfection method was used to transfect EGFPand eF64L,E222G in one, 8-well chamber with CHOhIR cells. Fluorescencefrom the cells was checked at regular intervals after transfection asdescribed above. Images were taken from the same cell fields at eachinterval. Three different fields were observed for each plasmid. Themicroscope and camera settings were the same for each image. Optimalexposure time was taken from a chamber of cells with full EGFPexpression (transfected 24 hours previously) to ensure the exposure doesnot saturate. The first images were taken from 45 minutes to 1 hour posttransfection, thereafter with a 30-minute interval for the first 7.5hours post transfection and an image was collected 26.5 hours posttransfection. Five different fields were observed for each plasmid.Fluorescence was detected no later then 4 hours post transfection.Fluorescence in eF64L,E222G was detected in one field 2.5 hours posttransfection. In the remaining fields, fluorescence was detected nolater than 4 hours post transfection (FIG. 4).

Example 4 Comparing pH Sensitivity Over Range pH 4.0 to pH 12.0 betweenEGFP and eF64L,E222G

[0094] Samples of semi-purified EGFP from PS279 and eF64L,E222G fromPS699 proteins produced in COS7 cell expression are tested for pHsensitivity over a range from pH 4.0 to pH 12.5, with 0.5 pointintervals. Excitation and emission scans were taken of each protein atthe pH values of 4.0, 8.0, and 12.5. The results of the scans foundEGFP's excitation max to be 490 nm and emission max to be 510 nm andeF64L,E222G 's excitation max to be 475 nm and emission max to be 504nm. Different pH values did not affect the excitation or emission max.Single reads were made with excitation at 470 nm, emission at 510 nm andwith 10 nm slits. The results show no clear differences between EGFP andeF64L,E222G regarding pH sensitivity, except what could be due to randomfluctuation (FIG. 5). This experiment has been repeated with essentiallysame result.

Example 5 Comparison of Relative Brightness of GFPs

[0095] 10 plasmids were constructed which combine some of the followingfeatures:

[0096] F or L at position 64.

[0097] S or T at position 65.

[0098] E or G at position 222.

[0099] “jellyfish” or “humanised enhanced” GFP backbone.

[0100] The plasmids were transfected into CHO cells. One, two and fourdays later the cells were inspected visually in a fluorescencemicroscope by two people. The excitation was 475/40=blue light and theemission 510-560=green light. Cells were scored on a “green” scaleranging from essentially black to extremely bright (Table 3). Resultsdid not change much with time. TABLE 3 codon aa aa aa Plasmid“greenness” GFP (* UVmax) context 64 65 222 PS854 black jf-GFP *jellyfish F S E PS845 almost black jf-GFP-E222G jellyfish F S G PS846almost black e-GFP-E222G humanised F S G PS844 almost black e-GFP *humanised F S E PS350 light green jf-GFP-F64L * jellyfish L S E PS351green jf-GFP-S65T jellyfish L T E PS832 green jf-GFP- jellyfish L S GF64L,E222G PS399 bright green e-GFP-F64L * humanised L S E PS699 verybright e-GFP- humanised L S G green F64L,E222G PS279 very bright EGFPhumanised L T E green

[0101] The plasmids were also transfected into HeLa cells. After 24hours transfection the cells were run on a FACS Calibur flow cytometerfor characterisation of whole cell fluorescence, with excitation at 488nm and emission viewed with fluorescence filter set 530/30 nm (range515-545 nm). 10000 events were collected for each transfection and 2replicates carried out for each construct. Average fluorescentintensities from the FACS analysis were obtained as geometric means(mean fluorescence on log scale) and results are shown in Table 4. TABLE4 GFP codon Plasmid FACS (* UVmax) context aa 64 aa 65 aa 222 PS845 5.4jf-GFP-E222G jellyfish F S G PS854 5.5 jf-wtGFP * jellyfish F S E PS3509.3 jf-BioGreen * jellyfish L S E PS846 9.4 e-wtGFP- humanised F S GE222G PS832 16.5 jf-BioE222G jellyfish L S G PS351 22.2 jf-BioSTjellyfish L T E PS844 24.5 e-wtGFP * humanised F S E PS399 73.3e-BioGreen * humanised L S E PS699 209.2 e-BioE222G humanised L S GPS279 421 EGFP humanised L T E

[0102] It is clear from the table above that, when expressed in themammalian HeLa cell, the GFPs with humanised codon are far brighter thanthe GFPs with jellyfish codon. EGFP and e-BioE222G being the brightest.It is no surprise that EGFP is about twice as bright as E-BioE222G underthese conditions. The excitation at the FACS is at 488 nm, close theexcitation maximum of EGFP at 490 nm. As illustrated in Table 5 below97% of the emission from EGFP will be picked up, whereas only 86% fromthe e-BioE222G. Furthermore, the difference between the intensity ofEGFP and e-bioE222G when excited at the e-bioE222G excitation maximum of470 is not as pronounced. TABLE 5 PS1189 PS1191 PS1185 PS1186 eLTE eLSGjfLTE jfLSG Emission intensity with excitation 131.4 94.1 155.0 167.2 at470 nm Emission intensity with excitation 148.1 80.4 178.2 151.2 at 488nm Excitation max 492 nm 468 nm 490 nm 473 nm Emission intensity atexcitation 152.9 93.8 183.3 169.1 max Ratio: Em. intensity(488)/Em. 0.970.86 0.97 0.89 intensity(max) Emission max 509 nm 505 nm 510 nm 506 nmEmission intensity at emission max 71.2 55.6 444 432

[0103] In mammalian cells enhanced GFPs were brighter than jellyfishGFPs. In E.Coli. jellyfish GFPs were brighter than enhanced GFPs. Thus,when it is worthwhile to choose the GFP backbone with care according tothe subsequent host.

1 24 1 720 DNA Aequoria Victoria CDS (1)...(717) 1 atg gtg agc aag ggcgag gag ctg ttc acc ggg gtg gtg ccc atc ctg 48 Met Val Ser Lys Gly GluGlu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 gtc gag ctg gac ggcgac gta aac ggc cac aag ttc agc gtg tcc ggc 96 Val Glu Leu Asp Gly AspVal Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 gag ggc gag ggc gat gccacc tac ggc aag ctg acc ctg aag ttc atc 144 Glu Gly Glu Gly Asp Ala ThrTyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 tgc acc acc ggc aag ctg cccgtg ccc tgg ccc aca cta gtg acc acc 192 Cys Thr Thr Gly Lys Leu Pro ValPro Trp Pro Thr Leu Val Thr Thr 50 55 60 ctg tct tac ggc gtg cag tgc ttcagc cgc tac ccc gac cac atg aag 240 Leu Ser Tyr Gly Val Gln Cys Phe SerArg Tyr Pro Asp His Met Lys 65 70 75 80 cag cac gac ttc ttc aag tcc gccatg ccc gaa ggc tac gtc cag gag 288 Gln His Asp Phe Phe Lys Ser Ala MetPro Glu Gly Tyr Val Gln Glu 85 90 95 cgc acc atc ttc ttc aag gac gac ggcaac tac aag acc cgc gcc gag 336 Arg Thr Ile Phe Phe Lys Asp Asp Gly AsnTyr Lys Thr Arg Ala Glu 100 105 110 gtg aag ttc gag ggc gac acc ctg gtgaac cgc atc gag ctg aag ggc 384 Val Lys Phe Glu Gly Asp Thr Leu Val AsnArg Ile Glu Leu Lys Gly 115 120 125 atc gac ttc aag gag gac ggc aac atcctg ggg cac aag ctg gag tac 432 Ile Asp Phe Lys Glu Asp Gly Asn Ile LeuGly His Lys Leu Glu Tyr 130 135 140 aac tac aac agc cac aac gtc tat atcatg gcc gac aag cag aag aac 480 Asn Tyr Asn Ser His Asn Val Tyr Ile MetAla Asp Lys Gln Lys Asn 145 150 155 160 ggc atc aag gtg aac ttc aag atccgc cac aac atc gag gac ggc agc 528 Gly Ile Lys Val Asn Phe Lys Ile ArgHis Asn Ile Glu Asp Gly Ser 165 170 175 gtg cag ctc gcc gac cac tac cagcag aac acc ccc atc ggc gac ggc 576 Val Gln Leu Ala Asp His Tyr Gln GlnAsn Thr Pro Ile Gly Asp Gly 180 185 190 ccc gtg ctg ctg ccc gac aac cactac ctg agc acc cag tcc gcc ctg 624 Pro Val Leu Leu Pro Asp Asn His TyrLeu Ser Thr Gln Ser Ala Leu 195 200 205 agc aaa gac ccc aac gag aag cgcgat cac atg gtc ctg ctg gag ttc 672 Ser Lys Asp Pro Asn Glu Lys Arg AspHis Met Val Leu Leu Glu Phe 210 215 220 gtg acc gcc gcc ggg atc act ctcggc atg gac gag ctg tac aag 717 Val Thr Ala Ala Gly Ile Thr Leu Gly MetAsp Glu Leu Tyr Lys 225 230 235 taa 720 2 239 PRT Aequoria Victoria 2Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 1015 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 2530 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 4045 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 5560 Leu Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 7075 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 8590 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu LysGly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys LeuGlu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp LysGln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His AsnIle Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln AsnThr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His TyrLeu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys ArgAsp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile ThrLeu Gly Met Asp Glu Leu Tyr Lys 225 230 235 3 720 DNA Aequoria VictoriaCDS (1)...(717) 3 atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtgccc atc ctg 48 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val ProIle Leu 1 5 10 15 gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agcgtg tcc ggc 96 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser ValSer Gly 20 25 30 gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aagttc atc 144 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys PheIle 35 40 45 tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc aca cta gtg accacc 192 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr50 55 60 ctg tct tac ggc gtg cag tgc ttc agc cgc tac ccc gac cac atg aag240 Leu Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 6570 75 80 cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag288 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 8590 95 cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgc gcc gag336 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100105 110 gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag ggc384 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115120 125 atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag tac432 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130135 140 aac tac aac agc cac aac gtc tat atc atg gcc gac aag cag aag aac480 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145150 155 160 ggc atc aag gtg aac ttc aag atc cgc cac aac atc gag gac ggcagc 528 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser165 170 175 gtg cag ctc gcc gac cac tac cag cag aac acc ccc atc ggc gacggc 576 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly180 185 190 ccc gtg ctg ctg ccc gac aac cac tac ctg agc acc cag tcc gccctg 624 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu195 200 205 agc aaa gac ccc aac gag aag cgc gat cac atg gtc ctc cta gggttc 672 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Gly Phe210 215 220 gtg acc gcc gcc ggg atc act ctc ggc atg gac gag ctg tac aag717 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230235 taa 720 4 239 PRT Aequoria Victoria 4 Met Val Ser Lys Gly Glu GluLeu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly AspVal Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp AlaThr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys LeuPro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Ser Tyr Gly Val GlnCys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe PheLys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe PheLys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe GluGly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp PheLys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn TyrAsn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu GlyPhe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu TyrLys 225 230 235 5 717 DNA Aequoria Victoria CDS (1)...(714) 5 atg agtaaa gga gaa gaa ctt ttc act gga gtt gtc cca att ctt gtt 48 Met Ser LysGly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 gaa ttagat ggc gat gtt aat ggg caa aaa ttc tct gtt agt gga gag 96 Glu Leu AspGly Asp Val Asn Gly Gln Lys Phe Ser Val Ser Gly Glu 20 25 30 ggt gaa ggtgat gca aca tac gga aaa ctt acc ctt aaa ttt att tgc 144 Gly Glu Gly AspAla Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 act act ggg aagcta cct gtt cca tgg cca acg ctt gtc act act ctc 192 Thr Thr Gly Lys LeuPro Val Pro Trp Pro Thr Leu Val Thr Thr Leu 50 55 60 tct tat ggt gtt caatgc ttt tct aga tac cca gat cat atg aaa cag 240 Ser Tyr Gly Val Gln CysPhe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 cat gac ttt ttc aagagt gcc atg ccc gaa ggt tat gta cag gaa aga 288 His Asp Phe Phe Lys SerAla Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 act ata ttt tac aaa gatgac ggg aac tac aag aca cgt gct gaa gtc 336 Thr Ile Phe Tyr Lys Asp AspGly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 aag ttt gaa ggt gat accctt gtt aat aga atc gag tta aaa ggt att 384 Lys Phe Glu Gly Asp Thr LeuVal Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 gat ttt aaa gaa gat ggaaac att ctt gga cac aaa atg gaa tac aat 432 Asp Phe Lys Glu Asp Gly AsnIle Leu Gly His Lys Met Glu Tyr Asn 130 135 140 tat aac tca cat aat gtatac atc atg gca gac aaa cca aag aat ggc 480 Tyr Asn Ser His Asn Val TyrIle Met Ala Asp Lys Pro Lys Asn Gly 145 150 155 160 atc aaa gtt aac ttcaaa att aga cac aac att aaa gat gga agc gtt 528 Ile Lys Val Asn Phe LysIle Arg His Asn Ile Lys Asp Gly Ser Val 165 170 175 caa tta gca gac cattat caa caa aat act cca att ggc gat ggc cct 576 Gln Leu Ala Asp His TyrGln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 gtc ctt tta cca gacaac cat tac ctg tcc acg caa tct gcc ctt tcc 624 Val Leu Leu Pro Asp AsnHis Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205 aaa gat ccc aac gaaaag aga gat cac atg atc ctt ctt gag ttt gta 672 Lys Asp Pro Asn Glu LysArg Asp His Met Ile Leu Leu Glu Phe Val 210 215 220 aca gct gct ggg attaca cat ggc atg gat gaa ggg tac aag 714 Thr Ala Ala Gly Ile Thr His GlyMet Asp Glu Gly Tyr Lys 225 230 235 taa 717 6 238 PRT Aequoria Victoria6 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 1015 Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe Ser Val Ser Gly Glu 20 2530 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 4045 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu 50 5560 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 7075 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 8590 95 Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys GlyIle 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Met GluTyr Asn 130 135 140 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys ProLys Asn Gly 145 150 155 160 Ile Lys Val Asn Phe Lys Ile Arg His Asn IleLys Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn ThrPro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr LeuSer Thr Gln Ser Ala Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg AspHis Met Ile Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr HisGly Met Asp Glu Gly Tyr Lys 225 230 235 7 717 DNA Aequovia Victoria CDS(1)...(717) 7 atg agt aaa gga gaa gaa ctt ttc act gga gtt gtc cca attctt gtt 48 Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile LeuVal 1 5 10 15 gaa tta gat ggc gat gtt aat ggg caa aaa ttc tct gtt agtgga gag 96 Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe Ser Val Ser GlyGlu 20 25 30 ggt gaa ggt gat gca aca tac gga aaa ctt acc ctt aaa ttt atttgc 144 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys35 40 45 act act ggg aag cta cct gtt cca tgg cca acg ctt gtc act act ctc192 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu 5055 60 tct tat ggt gtt caa tgc ttt tct aga tac cca gat cat atg aaa cag240 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 6570 75 80 cat gac ttt ttc aag agt gcc atg ccc gaa ggt tat gta cag gaa aga288 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 8590 95 act ata ttt tac aaa gat gac ggg aac tac aag aca cgt gct gaa gtc336 Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100105 110 aag ttt gaa ggt gat acc ctt gtt aat aga atc gag tta aaa ggt att384 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115120 125 gat ttt aaa gaa gat gga aac att ctt gga cac aaa atg gaa tac aat432 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Met Glu Tyr Asn 130135 140 tat aac tca cat aat gta tac atc atg gca gac aaa cca aag aat ggc480 Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Pro Lys Asn Gly 145150 155 160 atc aaa gtt aac ttc aaa att aga cac aac att aaa gat gga agcgtt 528 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val165 170 175 caa tta gca gac cat tat caa caa aat act cca att ggc gat ggccct 576 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro180 185 190 gtc ctt tta cca gac aac cat tac ctg tcc acg caa tct gcc ctttcc 624 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser195 200 205 aaa gat ccc aac gaa aag aga gat cac atg atc ctc cta ggg tttgta 672 Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu Leu Gly Phe Val210 215 220 aca gct gct ggg att aca cat ggc atg gat gaa cta tac aaa taa717 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys * 225 230235 8 238 PRT Aequovia Victoria 8 Met Ser Lys Gly Glu Glu Leu Phe ThrGly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn GlyGln Lys Phe Ser Val Ser Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Tyr GlyLys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val ProTrp Pro Thr Leu Val Thr Thr Leu 50 55 60 Ser Tyr Gly Val Gln Cys Phe SerArg Tyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys Ser AlaMet Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Phe Tyr Lys Asp AspGly Asn Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp ThrLeu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu AspGly Asn Ile Leu Gly His Lys Met Glu Tyr Asn 130 135 140 Tyr Asn Ser HisAsn Val Tyr Ile Met Ala Asp Lys Pro Lys Asn Gly 145 150 155 160 Ile LysVal Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val 165 170 175 GlnLeu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu Leu Gly Phe Val 210215 220 Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225 230235 9 33 DNA Artificial Sequence PCR Primer 9 tgtactagtg accaccctgtcttacggcgt gca 33 10 30 DNA Artificial Sequence PCR Primer 10 ctgactagtgtgggccaggg cacgggcagc 30 11 42 DNA Artificial Sequence PCR Primer 11cccggcggcg gtcacgaacc ctaggaggac catgtgatcg cg 42 12 42 DNA ArtificialSequence PCR Primer 12 cgcgatcaca tggtcctcct agggttcgtg accgccgccg gg 4213 31 DNA Artificial Sequence PCR Primer 13 gtaccggtca ccatgagtaaaggagaagaa c 31 14 29 DNA Artificial Sequence PCR Primer 14 ttattggtacccttcatcca tgccatgtg 29 15 41 DNA Artificial Sequence PCR Primer 15gagatcacat gatcctccta gggtttgtaa cagctgctgg g 41 16 41 DNA ArtificialSequence PCR Primer 16 cccagcagct gttacaaacc ctaggaggat catgtgatct c 4117 34 DNA Artificial Sequence PCR Primer 17 ccaacgcttg tcacaacgttttcttatggt gttc 34 18 34 DNA Artificial Sequence PCR Primer 18gaacaccata agaaaacgtt gtgacaagcg ttgg 34 19 34 DNA Artificial SequencePCR Primer 19 cccacactag tgacaacgtt ttcttacggc gtgc 34 20 34 DNAArtificial Sequence PCR Primer 20 gcacgccgta agaaaacgtt gtcactagtg tggg34 21 32 DNA Artificial Sequence PCR Primer 21 gttgtttcat gagtaaaggagaagaacttt tc 32 22 31 DNA Artificial Sequence PCR Primer 22 gttggatccttatttgtata gttcatccat g 31 23 32 DNA Artificial Sequence PCR Primer 23gttgttccat ggtgagcaag ggcgaggagc tg 32 24 31 DNA Artificial Sequence PCRPrimer 24 gttggatcct tacttgtaca gctcgtccat g 31

1. A fluorescent protein derived from Green Fluorescent Protein (GFP) orany functional GFP analogue, wherein the amino acid in position 1preceding the chromophore has been mutated and wherein the Glutamic acidin position 222 has been mutated said mutated GFP has an excitationmaximum at a higher wavelength and the fluorescence is increased whenthe mutated GFP is expressed in cells incubated at a temperature of 30°C. or above compared to wild-type GFP.
 2. A fluorescent proteinaccording claim 1, wherein the chromophore is in position 65-57 of thepredicted primary amino acid sequence of GFP.
 3. A fluorescent proteinaccording to claim 1 or 2, said protein being derived from Aequoriavictorea or Renilla.
 4. A fluorescent protein according to claim 1,wherein the amino acid F in position 64 of the GFP has been substitutedby an aliphatic amino acid.
 5. A fluorescent protein according to claim1, wherein the amino acid F in position 64 of the GFP has beensubstituted by an amino acid selected from the group consisting of L, I,V, A and G.
 6. A fluorescent protein according to claim 1, wherein theamino acid F in position 64 of the GFP has been substituted by L.
 7. Afluorescent protein according to claim 1, wherein the amino acid E inposition 222 of the GFP has been substituted by an amino acid selectedfrom the group consisting of G, A, V, L, I, F, S, T, N, and Q.
 8. Afluorescent protein according to claim 1, wherein the amino acid E inposition 222 of the GFP has been substituted by G.
 9. A fluorescentprotein according to claim 1 having the amino acid sequence disclosed inSEQ ID NO:
 4. 10. A fluorescent protein according to claim 1 having theamino acid sequence disclosed in SEQ ID NO:
 8. 11. A fusion compoundcomprising a fluorescent protein (GFP) according to claim 1, wherein theGFP is linked to a polypeptide.
 12. A fusion compound according to claim11, wherein the polypeptide is a kinase, preferably the catalyticsubunit of protein kinase A, or protein kinase C, or Erk1, or acytoskeletal element.
 13. A nucleotide sequence coding for thefluorescent protein of claim
 1. 14. A nucleotide sequence according toclaim 13, shown in SEQ ID NO:
 3. 15. A nucleotide sequence according toclaim 14, shown in SEQ ID NO:
 7. 16. A nucleotide sequence according toclaim 13 in the form of a DNA sequence.
 17. A host transformed with aDNA construct according to any one of claims 13-16.
 18. A process forpreparing a polypeptide, comprising cultivating a host according claim17 and obtaining therefrom the polypeptide expressed by said nucleotidesequence.
 19. A method for measuring the protein kinase activity,dephosphorylation activity or protein distribution in an in vitro assaycomprised of transforming a host cell with a DNA construct according toclaim 13 and measuring the fluorescence of cells transformed with theDNA construct.